INrf2:Nrf2 acts as a sensor for oxidative/electrophilic stress. INrf2 functions as an adaptor that interacts with Nrf2 and brings closer to ubiquitin ligase Cul3-Rbx1 for ubiquitination and degrading of Nrf2. Under basal conditions, INrf2/Cul3-Rbx1 complex is constantly degrading Nrf2. Nrf2 dissociates from the INrf2 in response to antioxidant and other stressors and translocates into the nucleus leading to activation of a myriad of genes that protect cells against stress and neoplasia. Immunoprecipitation and mass spectra analysis demonstrated interaction of INrf2 also with Phosphoglyerate mutase family member 5 (PGAM5)/BclXL/Bcl2, heat shock HSP90 and methylosome subunit pICln for unknown functions. Mass spectra analysis of immunoprecipitated INrf2 from antioxidant untreated and treated cells demonstrated phosphorylation of specific serine, threonine and tyrosine residues that might control INrf2 functions but remains unknown. Antioxidant treatment of cells led to genestein sensitive nuclear export of INrf2, Cul3 and Rbx1 presumably to allow nuclear import of Nrf2 without threat of degradation. However, the mechanism of export remains obscure. A feedback auto-regulatory loop between Nrf2 and INrf2 controls their cellular abundance. Interestingly, the Cul3 and Rbx1 gene expression is coordinately induced with INrf2 in response to antioxidant raising questions if Cul3 and Rbx1 are part of the INrf2-Nrf2 autoragulatory loop. The major goals of this proposal are to study role and regulation of INrf2. To this effect, we propose four specific aims. Aim 1 will investigate INrf2 interaction with PGAM5/BclXL/Bcl2, HSP90, and pICln and its significance in cellular protection and cell survival/death. The domain deletion, transfection, immunoprecipitation, Western analysis, reporter and apoptosis assays will be used in these studies. Aim 2 will elucidate the role of phosphorylation in control of homodimerization and stability of INrf2, nuclear localization, interaction with Cul3-Rbx1 and Nrf2 and degradation of Nrf2, antioxidant-induced release of Nrf2 from INrf2 and activation of gene expression, and interaction of INrf2 with PGAM5/BclXL/Bcl2, HSP90 and pICln. Sitedirected mutagenesis along with transfection and reporter assays, immunoprecipitation and Western analysis will be used to achieve the goals of this aim. Aim 3 will continue with generation of INrf2 conditional knockout mice that do not express INrf2 protein in liver and epidermis of skin. The Cre-Lox system will be used to generate these mice. In addition, compare the sensitivity of conditional INrf2 knockout mice expressing the null genotype with that of mice expressing INrf2, following carcinogen exposure, to develop skin and liver tumors. Aim 4 will test the hypothesis that Cul3 and Rbx1 are part of the auto-regulatory loop between INrf2 and Nrf2. Deletion mapping/transfection and band/super shift/ChIP assays will determine that ARE element and Nrf2 that regulate INrf2 also regulates antioxidant induction of Cul3 and Rbx1 genes. Co-transfection and ubiquitination assays will elucidate the role of coordinated induction of Cul3-Rbx1 in ubiquitination and degradation of Nrf2.